Identification, structural, and biophysical characterization of a positive modulator of human Kv3.1 channels

Figures

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  Fig. 1.. Discovery of the Kv3.1 potentiator and its biophysical properties. (A) Compound optimization scheme. Compound-1 was identified through library screening and further optimized by improving potency and solubility. (B) Concentration–response curves for compound-4 as measured in an automated whole-cell patch clamp assay performed in cells expressing Kv3.1 (Left) and Kv3.2 (Right) (mean ± SD, n = 3). The data points shown in red correspond to the concentrations at which compound-4 appeared to have either a less potentiating or an inhibitory effect on the channel. Compound effect (%) was determined as a change in the current amplitude measured in the presence of compound relative to the control current amplitude measured in the absence of compound-4. (C) Voltage-dependent activation of Kv3.1 channels. Patch clamp experiments were performed in a manual patch clamp assay from a holding potential of −100 mV to test potentials from −60 to +50 mV in step increments of +5 mV for 200 ms. The individual current traces demonstrate the increase in the Kv3.1 current at +10 mV after 1 μM compound-4 addition compared to the control. (D) Normalized current–voltage (Left) and conductance–voltage…

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  Fig. 2.. Overall structure of Kv3.1 in apo and potentiator-bound form, location of the binding pocket. (A) Density maps for apo and ligand-bound structures with one of the protomers highlighted using color-coding for different domains (VSD—green, PD—blue, T1—cyan). The density corresponding to the ligand is shown in yellow. (B) Overall structure of the apo and compound-bound Kv3.1 in two orientations (Top—along the membrane; Bottom—Top view from the extracellular side). The transmembrane helices of Kv3.1 are shown as purple cylinders, the T1 helices are shown as teal cylinders, the putative sterol molecule (CRL) is shown as green spheres, compound-4 (C4) is shown as orange spheres, the putative phospholipid (PL) in compound-4-bound structure is shown as green sticks, and the K+ and Zn2+ ions are shown as blue and red spheres. The turret domain is shown as a blue line and S1 to S2 loop is shown as a cyan line. The gray horizontal lines mark the membrane boundaries (EC—extracellular side, IC—intracellular side). (C) Overlay of T1 domain structures. Left: Apo structure (green) vs. compound-4-bound (magenta); Right: Our apo structure (green) vs. previously published apo structure (blue, PDB…

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  Fig. 3.. Potentiator binding pocket. (A) Interactions of compound-4 with its binding pocket. Compound-4 is shown as orange sticks, the phospholipid is shown as magenta sticks, and S4 and S5 helices are shown as pink cartoon with the relevant residues of the binding pocket shown as pink sticks and the hydrogen bonds between compound-4 and the protein backbone shown as red dashed lines. (B) Protein–ligand interaction diagram, highlighting the H-bonds (dashed arrows) with G370, A371, interactions with hydrophobic residues (green circles), and polar residues (purple circles). Blue circles around the ligand atoms (gradient color) and protein residues (solid color) indicate relative solvent exposure. (C) Densities around cholesterol in the apo structure and compound-4 contoured at 4 σ. (D) Sequence alignment of Kv3.1 with the rest of Kv3 subtypes and several ion channels of the other subfamilies. The residues lining the binding pocket are outlined by black rectangles.

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  Fig. 4.. Structural changes in Kv3.1 induced by potentiator binding. (A) Cartoon representation of Kv3.1 apo structure (green cartoon) overlayed with the compound-4-bound structure (magenta cartoon) with the extracellular loops of the ligand-bound structure colored for contrast as follows: S1 to S2 loop in teal, S3 to S4 in magenta, and the turret domain of S5 to S6 in blue. Compound-4 is shown in orange stick representation. Arrows indicate some of the larger-scale structural changes in Kv3.1 that occur upon compound-4 binding. (B) Close-up view at the turret domain and the residues contributing to the stability of the turret domain conformations in the apo and compound-bound states. The residue side chains of the apo structure are shown as green sticks, the residue side chains of the ligand-bound structure are shown as magenta sticks, and compound-4 is shown as orange sticks. The turret domain is highlighted, while the rest of the protein structure is semitransparent. The residues that were probed by point mutations are marked in bold. (C) Structural rearrangements that occur in the vicinity of the histidine residues H381 and H383. The color scheme is the same as panel B. (D) Structural…

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  Fig. 5.. Mechanism of voltage potentiation by compound-4. Schematic representation of a single functional subunit of Kv3.1 composed of the VSD shown in light blue and the PD of the adjacent protomer shown in dark blue. The extracellular loops in the apo state are shown in light gray and in black for the modulator-bound state. “+” and “−” labels denote positive and negative charges respectively on the lipidic bilayer and the voltage sensor helix S4. The crucial residues for the interdomain interactions are shown as sticks and magenta circles with yellow dashed lines indicating hydrogen bonds. The labels of the neighboring Kv3.1 protomer are marked by a prime. Compound-4 is shown as an orange rectangle with interactions to the hydrophobic residues of S4 and S5 shown as curved lines. The teal arrow denotes the electrostatic force exerted on the voltage sensor S4 by the membrane potential in the absence of a small molecule potentiator that returns S4 into the inward state.